71 results about "D-amino acid oxidase" patented technology

The invention discloses an enzyme-chemocatalysis racemization removing preparation method for L-glufosinate-ammonium. According to the method, a one-pot reaction manner is adopted, under the molecular oxygen, immobilization D-amino acid oxidase catalyzes D-enantiomer in an enantioselectivity mode into 2-imino-4-(hydroxy methyl phosphonyl) butyric acid in a dehydrogenation mode, and palladium-ammonium formate catalyzes 2-imino -4-(hydroxy methyl phosphonyl) butyric acid into DL-glufosinate-ammonium in an in-situ reduction mode. Hydrogen peroxide produced in the process is efficiently decomposed into water and oxygen through catalase. Complete reacemization removing of DL-glufosinate-ammonium and efficient preparing of L-glufosinate-ammonium are achieved through biological oxidation-chemical reduction circulation. The method has the advantages that the process is simple, cost is low, environmental friendliness is achieved, and energy is saved. High-concentration DL-glufosinate-ammonium can be converted into L-glufosinate-ammonium. The yield is 90%, the optical purity of the product is 99%, and the method is suitable for industrial production of L-glufosinate-ammonium.

The invention discloses a method for preparing L-phosphinothricin through deracemization and by a biological enzyme method. According to the method, the L-phosphinothricin is obtained by taking D,L-phosphinothricin as a raw material and through catalysis by an enzyme catalysis system, and the enzyme catalysis system consists of D-amino acid oxidase, amino acid dehydrogenase and coenzyme regeneration systems. By the method, racemized D,L-phosphinothricin serves as the raw material, the D-phosphinothricin is oxidized into 2-carbonyl-4-(hydroxymethylphosphonyl)-butyric acid by the D-amino acid oxidase, and the L-phosphinothricin does not take part in the reaction and is completely retained; but the 2-carbonyl-4-(hydroxymethylphosphonyl)-butyric acid is catalytically reduced into the L-phosphinothricin by the amino acid dehydrogenase, so that in-situ deracemization of the D,L-phosphinothricin is realized, the obtained L-phosphinothricin does not contain other side products, the total yield of the products is more than or equal to 99 percent, and the optical purify can exceed 99 percent.

This invention relates to compounds, compositions, and methods useful for modulating G72 and / or D-amino acid oxidase (DAAO) gene expression using short interfering nucleic acid (siNA) molecules. This invention also relates to compounds, compositions, and methods useful for modulating the expression and activity of other genes involved in pathways of G72 and / or D-amino acid oxidase (DAAO) gene expression and / or activity by RNA interference (RNAi) using small nucleic acid molecules. In particular, the instant invention features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methods used to modulate the expression of G72 and / or DAAO genes. The small nucleic acid molecules are useful in the treatment of schizophrenia and any other condition that responds to modulation of G72 and / or DAAO expression or activity.

The invention relates to recombinant microorganisms which, in comparison to the starting organism, have a higher concentration or activity of a D-amino acid oxidase, of an L-amino acid dehydrogenase, of an NADH cosubstrate regenerating enzyme and, if necessary, of a catalase. The invention also includes methods for preparing L-amino acids from D-amino acids using of these microorganisms.

Process for producing the enzyme D-amino acid oxydase of Rhodotorula gracilis in host cells. The process for the expression of the enzyme comprises isolating the complementary DNA corresponding to the messager RNA of the gene which codes for the D-amino acid oxydase of any strain of Rhodotorula gracillis producing said enzyme; fusing the fragment of DNA which codes for D-amino acid oxydase of Rhodotorula gracillis with a DNA sequence which contains a site of union to the ribosome and a high efficiency promoter sequence for the express of genes in host cells; inserting the DNA fragment into a plasmid appropriate for the host cell; cultivating the host cells transformed with said plasmid; and collecting the enzyme.

The invention discloses fructose amino acid oxidase which has an amino acid sequence shown as SEQID.No.1 (sequence identifier number 1) or has above 80% homology with the amino acid sequence. One or more amino acid residues in corresponding positions of amino acid selected from (a) to (f) are substituted. The obtained fructose amino acid oxidase has higher thermostability: (a) 59-site glutamic acid, (b) 98-site glutamic acid, (c) 225-site glycine, (d) 277-site lysine, (e) 283-site glutamic acid and (f) 355-site aspartic acid. The invention further discloses a preparation method of the oxidase and a kit comprising the oxidase and used for determining glycatedalbumin. The kit has higher thermostability and can accurately determine the glycatedalbumin.

The invention relates to an enzymatic measuring method of glycosylated serum protein and a relative liquid stabilizer, with wide application in medical and biochemical technical field. The invention is characterized in that glycosylated serum protein is digested by bromelain to generate fructose amino acid, fructose amino acid oxidase is reacted with the fructose amino acid to generate H2O2 to be reacted with chromogen DA-64 in the presence of peroxidase to convert DA-64 into one green product, the product content is in direct proportion with glycosylated serum protein content of sample, and an automatic biochemical analyzer uses two-point end point method to measure the content of glycosylated serum protein in the sample. The inventive liquid measuring agent has high stability, easy application, strong interference resistance and non pollution on the pipeline of automatic biochemical analyzer, which is suitable for automatically measuring glycosylated serum protein in batch.

The invention discloses a mutant zymoprotein of D-amino acid oxidase and preparation method of the mutant zymoprotein. The preparation method comprises the following steps: according to homologous sequence comparison result, respectively performing mutation on 115, 119 and 286 sites of D-amino acid oxidase in primary Arthrobacterprotophormiae (DSM15035) through site-specific mutagenesis technology to construct three-site mutation expression vector Pet-e115A / N119D / T286A; and carrying out prokaryotic expression and purification to obtain zymoprotein. An apparent secondary speed constant Kcat / Km of the obtained mutant protein to substrate D-Met is 1.39*10 to the power of 5s-1.M-1, is 5.03 times of wild type DAAO (D amino acid oxidase), and is 55.96 times of pKDAAO protein of pig kidney source. The detection efficiency of partial D-amino acid can be improved, and the good application value is realized.

The invention discloses a method for preparing L-glufosinate-ammonium through microorganism catalysis and deracemization. According to the method, DL-glufosinate-ammonium is used as a raw material, D-amino acid oxidase in lysinibacillus xylanilyticus XX-2 whole cells catalyzes D-glufosinate-ammonium to be subjected to oxidative deamination to obtain 4-(hydroxymethylphosphinyl)-2-oxobutanoic, and L-glufosinate-ammonium is reserved. The amino acid dehydrogenase co-expressed in cells catalyzes the 4-(hydroxymethylphosphinyl)-2-oxobutanoic to be subjected to in-situ reduction and ammoniation to obtain the L-glufosinate-ammonium, so that complete deracemization of the DL-glufosinate-ammonium is realized. The prepared L-glufosinate-ammonium does not have any other by-products, the total yield isgreater than 70%, and the optical purity is greater than 99%.

The invention is a method of preparing D-amino acid oxidase, relating to a method of preparing flavo-enzyme D-amino acid oxidase. Concretely, it relates to a method of highly efficiently producing mutational D-amino acid oxidase by constructing recombinant engineering strains. It reconstructs wild D-amino acid oxidase coming from Trigonopsis variablilis by means of gene engineering, fuses a segment of Histag at N end and C end of the wild enzyme, and further implements high-efficiency fast separation of mutational enzyme by affinity chromatography. The expression level of the enzyme in fermentation liquor is 4000IU / mL, higher than that of the wild enzyme, and the recovery ratio of Ni-column affinity chromatography is 50%. The purified enzyme can be used in making high-efficient oxidation conversion of cephalosporin C- to produce glutaryl-7-ACA and be used together with GL-7-ACA to produce 7-ACA.

The invention discloses a glufosinate-ammonium dehydrogenase mutant and an application thereof to production of L-glufosinate-ammonium through oxidation-reduction of multienzymes. According to the method, a method for preparing an L-glufosinate-ammonium precursor through D-amino acid oxidase is utilized, D, L-glufosinate-ammonium or D-glufosinate-ammonium is used as substrate, and in aerobic environment or under the situation that catalase exists, through excised D-amino acid oxidase or in vitro expression of cells of D-amino acid oxidase, the D-glufosinate-ammonium is catalyzed to obtain 4-(hydroxymethylphosphinyl)-2-oxobutanoic; and under the action of glufosinate-ammonium dehydrogenase, the 4-(hydroxymethylphosphinyl)-2-oxobutanoic is catalyzed to generate the L-glufosinate-ammonium, the activity of a catalyst is improved by about 10 times, and the concentration of the substrate is increased by 5 times. The method is high in raw material conversion rate and high in yield, and products are easy to separate and purify.

The invention concerns the human DAO gene, polynucleotides, and biallelic markers. The invention also concerns the association established between schizophrenia and the biallelic markers. The invention provides means to determine the predisposition of individuals to schizophrenia or related CNS disorder, as well as means for the disease diagnosis and prognosis.

The invention provides Escherichia coli expression vector and its using method. The expression vector is formed by vector pRSET-A whose ampicillin resistant gene is replaced by kanamycin resistant gene. And it can be used to express the enzyme needed in making 7-aminocephalosporanic acid but no beta-lactamase reduced its yield. The invention also provides two concrete expression vectors that pHS-GHA and pT7-kan-ACY whose gene products are respectively D-amino acid oxidase mutant GHA and Pseudomonas sp.SE83 glutaryl-7-aminocephalosporanic acid acidylating enzyme.

A composition of variant biocatalysts, specifically variants of D-amino acid oxidases, with improved biocatalytic activity towards D-amino acid substrates such as, but not limited to, D-tert-leucine, D-norvaline, D-2-aminobutyrate, D-alanine, D-isoleucine, D-valine, D-methionine, D-hydroxyadamantlyglycine, D-penicillamine, or D-norleucine is disclosed. Further disclosed is a method of preparing enantioselective amino acids using variant D-amino acid oxidases of the present invention.

The invention discloses a gene complete sequence of siganus oramin L-amino acid oxidase and application thereof. The gene complete sequence of the siganus oramin L-amino acid oxidase is 1725bp, the length of an open reading frame is 1584bp, and the gene complete sequence is specifically in a 43-1626 region and is used for coding 527 amino acids. The siganus oramin L-amino acid oxidase is obtained from siganus oramin serum through separation and purification, and has a function of killing cryptocaryon irritans brown and a bactericidal activity on typical gram-positive bacteria (staphylococcus aureus) and gram-negative bacteria (escherichia coli). In an optimized high-definition enzyme PCR (Polymerase Chain Reaction) system, by using total siganus oramin spleen cDNA as a template and adopting a pair of designed specific primers, the gene sequence coding the protein can be rapidly accurately cloned and used for the subsequent genetic engineering.

The invention discloses a method and a kit for detecting concentration of chiral amino acid. The method comprises the following steps of: 1) adding D-amino acid oxidase or L-amino acid oxidase into a sample to be tested for carrying out an enzyme catalysis reaction; 2) sequentially adding buffer solution, luminol and horse radish peroxidase into the obtained reaction solution in the step 1) for carrying out a chemiluminiscence reaction; and 3) measuring the luminous intensity of the reaction solution in the step 2). The method can be used for measuring chiral amino acid in an enantiomer. The method is not interfered by another chiral amino acid in the enantiomer or is not interfered by common metal ions. The method is easy to operate and high in sensitivity, and the content of the chiral amino acid in the enantiomer can be detected.

Provided is a compound having a D-amino acid oxidase (DAAO) inhibitory action, and useful as for example, a prophylaxis and / or therapeutic agent for schizophrenia or neuropathic pain. The present inventors have studied a compound that inhibits DAAO, and confirm that a dihydroxy aromatic heterocyclic compound has a DAAO inhibitory action, and completed the present invention. That is, the dihydroxy aromatic heterocyclic compound of the present invention has a good DAAO inhibitory action, and can be used as a prophylaxis and / or therapeutic agent for, for example, schizophrenia or neuropathic pain.

A method for purification of cephalosporin C broth using absorbent is provided, thereby effectively removing diacetyl CPC(cephalosporin C) from the cultured medium. The method for purification of cephalosporin C broth using absorbent comprises the steps of: filtering and absorbing cephalosporin C(CPC) cultured medium using absorbent such as polystyrene resin, and eluting it to remove diacetyl CPC from the CPC cultured medium; reacting cephalosporin C(CPC) cultured medium with D-amino acid oxidase(DAOD) to prepare glutaryl-7-aminocephalosporanic acid(Gl-7-ACA); and reacting glutaryl-7-aminocephalosporanic acid(Gl-7-ACA) with glutaryl-7-aminocephalosporanic acid acylase(Gl-7-ACA acylase) to prepare 7-aminocephalosporanic acid(7-ACA), wherein the CPC cultured medium has pH 5.0 to 6.0; and the eluting solution is phosphate buffer solution with pH 6 to 9, acetate buffer solution, carbonate buffer solution or weak alkali solution.

A recombinative D- amino acid oxidase and its DNA code sequence. Activity to catalysis rhzomorphC of the enzyme is 25% higher than that of its parents at least.

The invention discloses a mutant of L-amino acid oxidase and a preparation method and application thereof, and belongs to the technical field of gene engineering. Through performing site-specific mutagenesis on the L-amino acid oxidase derived from corynebacterium glutamicum, a mutant Q183A is obtained. Heterologous expression is performed on escherichia coli through the mutant Q183A performs, catalytic efficiency of obtained recombinant bacteria to L-valine is improved by 18.7%, a yield of alpha-ketoisocaproic acid reaches 54.6 g / L, and a molar conversion rate of a substrate is 84.6%. Compared with a chemical method, the provided whole-cell transformation method has the advantages of moderate reaction condition, single target product, easy separation, environment-friendliness and the like, and is simple in process, easy to control, and easy for popularization and application.

This invention provides a method of enhancing NMDAR-mediated neurotransmission in a disease associated with NMDAR antibody production, said method comprising administering an NMDAR agonist, an alanine-serine-cysteine transporter inhibitor, a D-amino acid oxidase inhibitor, a glycine transport inhibitor or a combination thereof to said subject. This invention also provides a method of mitigating the severity of, mitigating the pathogenesis of, lowering the incidence of or treating a disease associated with NMDAR antibody production, said method comprising administering an agent, which is an NMDAR agonist, an alanine-serine-cysteine transporter inhibitor, a D-amino acid oxidase inhibitor, a glycine transport inhibitor or a combination thereof to said subject.

Provided herein are compounds of Formula (I) and uses thereof for inhibiting the activity of D-amino acid oxidase (DAAO) or treating diseases or disorders associated with DAAO, such as a central nervous system (CNS) disorder, obesity, diabetes, or hyperlipidemia. Also provided in the present disclosure are methods of synthesizing the Formula (I) compounds described herein.

The invention discloses a method for quantitatively detecting the activity of L-amino acid oxidase by using a Prussian blue agar plate, which comprises the following steps: with a punch, punching a small hole having a diameter of 4-10 mm on the Prussian blue agar plate; then, adding a liquid to be determined into the small hole, and standing at room temperature for 10-30 minutes, wherein hydrogen peroxide generated by the liquid to be determined forms a blue ring around the small hole; and determining the diameter of the blue ring, determining the release amount of hydrogen peroxide according to the hydrogen peroxide and a standard straight line made of the diameter of the blue ring, and then deducing to obtain the activity of L-amino acid oxidase, wherein the liquid to be determined is a reaction liquid obtained by reacting an enzyme source and a substrate under the conditions of a temperature of 25-50 DEG C and a pH value of 5-9 for 0.5-2 hours, the enzyme source is an enzyme obtained through the fermentation culture of Pseudoalteromonas sp. B3, and the substrate is L-amino acid. The method has the characteristics of low cost, simple and convenient operation process, wide applicability, high stability and the like.

This invention provides novel inhibitors of the enzyme D-amino acid oxidase as well as pharmaceutical compositions including the compounds of the invention. Also provided are methods for the treatment and prevention of neurological disorders, such as neuropsychiatric and neurodegenerative diseases, as well as pain, ataxia and convulsion. The compounds of the invention have the general structure wherein Q is a member selected from O, S, CR and N, X and Y are members independently selected from CR, O, S, N and NR.

The preparation method of D-amino acid oxides is characterized by that is uses colibacillus gene engineering bacterium to make shake-flask strain culture in culture medium, fermentation strain culture and fermentation culture. Said invention is a preparation method which utilizes the colibacillus gene engineering bacterium to make fermentation production, utilizes centrifuge to collect thallus, soakes to extract enzyme liquor, and makes the enzyme liquor undergo the processes of plate-frame filtration, ultra filtration and concentration, purification and fixation so as to obtain the invented product.

The invention discloses a primary amine immobilized enzyme carrier and a preparation method thereof. Positive suspension polarization is performed to obtain a crude macroporous polymethacrylate-diallylamine carrier; washing the crude carrier, and grading; performing amination to obtain the primary amine-containing immobilized enzyme carrier. The primary amine immobilized enzyme carrier according to the technical scheme of the invention is applicable to immobilization of various biological enzymes, provides effective immobilization for penicillin G acylase, D-amino acid oxidase, Gl-7-ACA acylase and the like, and has high enzyme-carrying activity, good mechanical strength and good storage stability. The preparation method is simple and has low cost, and the carrier has significantly improved storage stability and is expectedly applicable to industrial large-scale production.

The present invention relates to compounds of Formula (I):or a pharmaceutically acceptable salt thereof, wherein: each of A, B, C, D, and E, independently, is C, N, N—H, O, S, or absent is a single bond or a double bond; each of X, Y, and Z, independently, is aryl, heteroaryl, aralkyl, H, or absent; each of L1 and L2, independently, is a moiety selected from O, CH2, C═O, C2-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, —((CH2)n—W)—, wherein n=0, 1, 2, 3, 4, or 5, and W is O or S, or absent; and when L2 is absent, Z is aryl or heteroaryl fused with BC. Also provided in the present invention is a method for inhibiting, treating and / or reducing the risk of a neuropsychiatric disorder, comprising administering a subject in need a composition comprising a compound of Formula (I).